1. Field of the Invention
The present invention relates to therapeutic and diagnostic methods and compositions based on Jagged/Notch proteins and nucleic acids, and on the role of their signaling pathway in endothelial cell migration and/or differentiation.
2. Background of the Invention
The functional integrity of the human vascular system is maintained by the endothelial cell which monitors the non-thrombogenic interface between blood and tissue in vivo. Thus, factors that influence human endothelial cell function may contribute significantly to the regulation and maintenance of homeostasis (see, Maciag, in Progress in Hemostasis and Thrombosis, T. Spaet, ed. (New York: A. R. Liss), pp.167-182 (1984); Folkman and Klagsbum, Science235:442-447 (1987); Burgess and Maciag, Annu. Rev. Biochen. 58:575-606 (1989)). Likewise, events that perturb this complex equilibrium are relevant to the pathophysiology of human disease states in which cellular components of the vascular tree are active participants including, e.g., atherogenesis, coronary insufficiency, hypertension, rheumatoid arthritis, solid tumor growth and metastasis, and wound repair.
Since the endothelium is present in all organs and tissues, endothelial cell function is also fundamental to the physiology and integration of these multicellular systems. This includes the ability to monitor and interface with repair systems that employ the tightly regulated inflammatory, angiogenic and neurotropic responses. Indeed, biochemical signals that are responsible for the modification of these responses have been well characterized as polypeptide growth factors and cytokines; however, their mechanisms of operation have, prior to the present invention, been poorly understood, impeding their acceptance as valuable tools in clinical management.
A major accomplishment of modern biology has been the recognition that structural elements responsible for physiologic functions are conserved throughout the animal kingdom. Genetic analysis of yeast, C. elegans, Xenopus, Zebra fish, and Drosophila, among others, have provided new insight into the regulation of the cell cycle, organelle biosynthesis and trafficking, cell fate and lineage decisions during development, as well as providing the fundamental principles for transcriptional/translational/post-translational regulation. Indeed, the conservation of structure-function principles exhibited by such systems has generated new insight into these and other regulatory systems utilized by mammalian cells. Moreover, a resolution of the genetic structure of the mammalian homologs for such genes in non-mammalian species has often led to a discernment of their function in mammals, even though the delineation of the function of a particular, homologous mammalian gene or gene fragment may well be serendipitous. In many cases, it is the result produced by expression and differential cDNA cloning strategies that manifest mammalian DNA sequences with homology to genes previously identified in more primitive species.
During the past decade, differential cDNA cloning methods, including e.g., conventional subtractive hybridization (Hla and Maciag, Biochem. Biophys. Res. Commun. 167:637-643 (1990a)), differential polymerase chain reaction (PCR)-oriented hybridization (Hla and Maciag, J. Biol. Chem. 265:9308-9313 (1990b)), and more recently, a modification of the differential display (Zimrin et al., Biochem. Biophys. Res. Commun. 213:630-638 (1995)) were used to identify genes induced during the process of human umbilical vein endothelial cell (HUVEC) differentiation in vitro. Very early studies disclosed that HUVEC populations are able to generate capillary-like, lumen-containing structures when introduced into a growth-limited environment in vitro (Maciag et al., J. Cell Biol. 94:511-520 (1982)). These studies permitted the identification and characterization of protein components of the extracellular matrix as inducers of this differentiation process, while at the same time defining the capillary-like structures as non-terminally differentiated (Maciag, 1984). Additional experiments have elucidated the importance of polypeptide cytokines, such as IL1 (Maier et al., J. Biol. Chem. 265:10805-10808 (1990a)), and IFNxcex3 (Friesel et al., J. Cell Biol. 104:689-696 (1987)) as inducers of HUVEC differentiation in vitro, and ultimately lead to an understanding that the precursor form of IL-1xcex1 was responsible for the induction of HUVEC senescence in vitro (Maciag et al., J. Cell Biol. 91:420-426 (1981); Maier et al., Science 249:1570-1574 (1990b))xe2x80x94the only truly terminal HUVEC phenotype identified to date. Summarized in FIG. 1.
Recent research has employed differential cDNA cloning methods, which permits the identification of new and very interesting genes. However, until very recently, establishing their identity did not provide insight into the mechanism of HUVEC differentiation. Current research has focused upon the fibroblast growth factor (FGF) and interleukin (IL)-1 gene families as regulators of the angiogenesis process, both in vitro and in vivo (Friesel et al., FASEB J9:919-925 (1995); Zimrin et al., J. Clin. Invest. 97:1359 (1996)). The human umbilical vein endothelial cell (HUVEC) has proven to be an effective model for studying the signal pathways utilized by FGF-1 to initiate HUVEC migration and growth, the role of IL-1xcex1 as an intracellular inhibitor of FGF-1 function and modifier of HUVEC senescence, and the interplay between the FGF and the IL-1 gene families as key effectors of HUVEC differentiation in vitro. Such insight has enabled the present inventors to use modern molecular methods to identify a key regulatory ligand-receptor signaling system, which is able to both induce capillary endothelial cell migration and repress large vessel endothelial cell migration.
The Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, originally described during the development of C. elegans and Drosophila as an essential system instrumental in cell fate decisions, has been found to be highly conserved in mammalian cells (Nye and Kopan, Curr. Biol. 5:966-969 (1995)). Notch proteins comprise a family of closely-related transmembrane receptors initially identified in embryologic studies in Drosophila (Fortini and Artavanis-Tsakonas, Cell 75:1245-1247 (1993)). The genes encoding the Notch receptor show a high degree of structural conservation, and contain multiple EGF repeats in their extracellular domains (Coffinan et al., Science 249:1438-1441 (1990); Ellisen et al., Cell 66:649-661 (1991); Weinmaster et al., Development 113:199-205 (1991); Weinmaster et al., Development 116:931-941 (1992); Franco del Amo et al., Development 115:73 7-744 (1992); Reaume et al., Dev. Biol. 154:377-387 (1992); Lardelli and Lendahi, Mech. Dev. 46:123-136 (1993); Bierkamp and Campos-Ortega, Mech. Dev. 43:87-100 (1993); Lardelli et al., Exp. Cell Res. 204:364-372 (1994)). In addition to the 36 EGF repeats within the extracellular domain of Notch 1, there is a cys-rich domain composed of three Notch Lin Glp (NLG) repeats, which is important for ligand function, followed by a cys-poor region between the transmembrane and NLG domain.
The intracellular domain of Notch 1 contains six ankyrin/Cdc10 repeats positioned between two nuclear localization sequences (NLS) (Artavanis-Tsakonas et al., Science 268:225-232 (1995)). This motif is found in many functionally diverse proteins (see e.g., Bork, Proteins 17:363-374 (1993)), including members of the rel/NF-kB family (Blank et al., TIBS 17:135-140 (1992)), and is thought to be responsible for protein-protein interactions. Notch has been shown to interact with a novel ubiquitously distributed cytoplasmic protein deltex through its ankyrin repeats, a domain shown by deletion analysis to be necessary for activity (Matsuno et al., Development 121:2633-2644 (1995)).
Carboxy terminal to this region is a polyglutamine-rich domain (OPA) and a pro-glu-ser-thr (PEST) domain which may be involved in signaling protein degradation. There are numerous Notch homologs, including three Notch genes. (The corresponding structures for Lin-12 and Glp-1 are shown in FIG. 2.)
Several Notch ligands have been identified in vertebrates, including Delta, Serrate and Jagged. The Notch ligands are also transmembrane proteins, having highly conserved structures. These ligands are known to signal cell fate and pattern formation decisions through the binding to the Lin-12/Notch family of transmembrane receptors Nuskavitch and Hoffmann, Curr. Top. Dev. Biol. 24:289-328 (1990); Artavanis-Tsakonas and Simpson, Trends Genet. 7:403408 (1991); Greenwald and Rubin, Cell 68:271-281 (1992); Gurdon, Cell 68:185-199 (1992); Fortini and Artavanis-Tsakonas, 1993; and Weintraub, Cell 75:1241-1244 (1993)). A related protein, the Suppressor of hairless (Su(H)), when co-expressed with Notch in Drosophila cells, is sequestered in the cytosol, but is translocated to the nucleus when Notch binds to its ligand Delta (Fortini and Artavanis-Tsakonas, 1993). Studies with constitutively activated Notch proteins missing their extracellular domains have shown that activated Notch suppresses neurogenic and mesodermal differentiation (Coffman et al., Cell 73:659-671 (1993); Nye et al., Development 120:2421-2430 (1994)).
The Notch signaling pathway (FIG. 3), which is apparently activated by Jagged in the endothelial cell, involves cleavage of the intracellular domain by a protease, followed by nuclear trafficking of the Notch fragment and the interaction of this fragment with the KBF2/RBP-Jk transcription factor (Jarriault et al., Nature 377:355-358 (1995); Kopan et al., Proc. Natl. Acad Sci. USA 93:1683-1688 (1996)), a homolog of the Drosophila Suppressor of hairless gene (Schweisguth et al., Cell 69:1199-1212 (1992)), a basic helix-loop-helix transcription factor involved in Notch signaling in insects (Jennings et al., Development 120:3537-3548 (1994)) and in the mouse (Sasai et al., Genes Dev. 6:2620-2634 (1992)). This effector is able to repress the transcriptional activity of other genes encoding transcription factors responsible for entry into the terminal differentiation program (Nye et al., 1994; Kopan et al., J. Cell. Physiol. 125:1-9 (1994)).
The Jagged gene encodes a transmembrane protein which is directed to the cell surface by the presence of a signal peptide sequence (Lindsell et al., Cell 80:909-917 (1995)). While the intracellular domain contains a sequence with no known homology to intracellular regions of other transmembrane structures, the extracellular region of the ligand contains a cys-rich region, 16 epidermal growth factor (EGF) repeats, and a DSL (Delta Serrate Lag) domain. As shown in FIG. 4, the DSL domain as well as the EGF repeats, are found in other genes including the Drosophila ligands, Serrate (Baker et al., Science 250:1370-13771990; Thomas et al., Development 111:749-761 (1991)) and Delta (Kopczynski et al., Genes Dev. 2:1723-1735 (1988)), and C. elegans genes Apx-1 (Henderson et al., Development 120:2913-2924 (1994); Mello et al., Cell 77:95-106 (1994)) and Lag-2 (Tax et al., Nature 368, 150-154 (1994)).
Nevertheless, until the discovery of the presently disclosed invention, human Jagged remained undefined and the function and relationship, if any, of the human ligand to Notch remained unknown in the art. However, there was a recognized need in the art for a complete understanding of the protein""s role in the regulation of cell differentiation and regulation. As disclosed in the present invention, the human Jagged gene has now been cloned, isolated and defined, and the Jagged-Notch role in endothelial cell differentiation and/or migration has been elucidated. In addition, it is presently disclosed that the novel signaling pathway produces disparate effects on the migration of large and small vessel endothelial cells, providing what appears to be the first demonstration of a signaling difference between large and small vessel endothelial cells both in degree and direction. This highlights the potential function of a previously unknown ligand-receptor signaling pathway in the endothelial cell which is modulated during the migratory phase of angiogenesis. Moreover, the present invention provides an explanation of the previously unresolved phenomenon in which endothelial cells have been shown to reproducibly differentiate into a non-terminal and completely reversible tubular-like cell phenotype in vitro (Maciag et al., 1982). Thus, the present invention significantly advances the art, providing not only methods of regulating cell differentiation and angiogenesis, but also teaching a method for preventing the undesirable migration of specific cell types into large blood vessels following angioplastic surgery to control restenosis.
The present invention relates to a novel discovery of human Jagged and of the role of Jagged-Notch in endothelial cell migration and/or differentiation, and to the determination that the signaling pathway produces disparate effects on the migration of large and small vessel endothelial cells.
The invention provides a substantially purified Jagged protein, ie., a peptide free of the proteins with which it is normally associated, particularly a human Jagged protein; it also provides a functionally equivalent derivative, or allelic or species variant thereof. It further provides a peptide which has an amino acid sequence corresponding to SEQ ID NO:1. Moreover, the invention provides a protein which is characterized by the ability to bind to Notch.
The invention provides a substantially purified nucleic acid molecule encoding a Jagged protein, particularly a human Jagged protein; it also provides a nucleic acid molecule or DNA segment thereof encoding a functionally equivalent derivative, or allelic or species variant thereof. It further provides a nucleic acid sequence having a sequence corresponding to SEQ ID NO:2. Moreover, the invention provides a nucleic acid sequence encoding a human protein which is characterized by the ability to bind to Notch.
In addition, the invention provides a recombinant molecule comprising a vector and the nucleic acid sequence or segment thereof encoding the Jagged protein or functional portion thereof, particularly the human Jagged protein. It also provide a host cell comprising the recombinant molecule comprising a vector and the nucleic acid sequence or segment thereof encoding the Jagged protein or functional portion thereof The invention further provides the expression product of the recombinant molecule comprising a vector and the nucleic acid sequence encoding the Jagged protein.
Further, the invention provides a substantially purified, single-stranded, nucleic acid molecule comprising the antisense strand of the Jagged cDNA (xcex3-Jagged), particularly of the cDNA for the human Jagged protein; it also provides DNA segments which if read in the sense direction would encode a functionally equivalent derivative, or allelic or species variant thereof. It also provides the nucleic acid molecule comprising the antisense nucleotide sequence corresponding to the antisense strand of SEQ ID NO:2. Moreover, the invention provides an antisense molecule which is characterized by the ability to bind to Jagged, or a functionally equivalent derivative, or allelic or species variant thereof.
The invention also provides the polypeptide encoded by the nucleic acid molecule comprising the antisense strand of the Jagged cDNA (xcex3-Jagged), particularly of the cDNA for the human Jagged protein. It further provides the polypeptide encoded by the antisense Jagged molecule, wherein the polypeptide has a binding affinity to, and inhibits the activity of Jagged.
In addition, the invention provides an antibody having a binding affinity to Jagged, or a unique portion thereof.
It also provides a secondary antibody having a binding affinity to anti-Jagged, or a unique portion thereof.
The invention provides a method of decreasing the migration of endothelial cells to a site on a micro-diameter blood vessel, comprising delivering a Jagged protein, or a functionally equivalent derivative, or allelic or species variant thereof, or a secondary anti-Jagged antibody to a site from which the endothelial cells have been removed, damaged or substantially reduced. It also provides a method of decreasing the migration of endothelial cells, particularly human endothelial cells, to a site on a macro-diameter blood vessel, comprising delivering an antisense Jagged molecule (xcex3-Jagged) or a Jagged antibody to a site from which the endothelial cells have been removed, damaged or substantially reduced.
The invention provides a method of increasing the migration of endothelial cells, particularly human endothelial cells, to a site on a macro-diameter blood vessel, comprising delivering a Jagged protein, or a functionally equivalent derivative, or allelic or species variant thereof or a secondary anti-Jagged antibody to a site from which the endothelial cells have been removed, damaged or substantially reduced. It also provides a method of increasing the migration of endothelial cells, particularly human endothelial cells, to a site on a micro-diameter blood vessel, comprising delivering an antisense Jagged molecule (xcex3-Jagged) or a Jagged antibody to a site from which the endothelial cells have been removed, damaged or substantially reduced.
Moreover, the invention provides a method of decreasing the migration of smooth muscle cells, particularly human smooth muscle cells, to a site on a macro-diameter blood vessel, comprising delivering an antisense Jagged molecule (xcex3-Jagged) or a Jagged antibody to a site from which the endothelial cells have been removed, damaged or substantially reduced.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a Jagged protein, or functionally equivalent derivative, or allelic or species variant thereof particularly a human Jagged protein; and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of a Jagged nucleic acid, or functionally equivalent derivative, or allelic or species variant thereof, particularly a human Jagged nucleic acid; and a pharmaceutically acceptable carrier.
In addition, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a Jagged antibody, or functionally equivalent derivative, or allelic or species variant thereof; and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of a Jagged antisense molecule, or functionally equivalent derivative, or allelic or species variant thereof; and a pharmaceutically acceptable carrier. Further provided is a pharmaceutical composition comprising a therapeutically effective amount of an anti-Jagged antibody, or functionally equivalent derivative, or allelic or species variant thereof; and a pharmaceutically acceptable carrier.
The invention also provides a method of preventing or treating a disease or condition in a subject comprising administering to a subject in need of such prevention or treatment a therapeutically effective amount of a molecule which antagonizes, inhibits or prevents the function of the Notch protein; or comprising administering a therapeutically effective amount of a molecule which agonizes, enhances or stimulates the function of the Notch protein. It further provides a method of preventing or treating a disease or condition in a subject comprising administering to a subject in need of such prevention or treatment a therapeutically effective amount of a molecule which antagonizes, inhibits or prevents the function of the Jagged protein; or comprising administering a therapeutically effective amount of a molecule which agonizes, enhances or stimulates the function of the Jagged protein.
In addition, the invention provides a method of inhibiting or preventing angiogenesis in a subject comprising administering to a subject in need of such inhibition or prevention a therapeutically effective amount of Jagged or a Jagged agonist. The angiogenesis being inhibited or prevented comprises solid tumor angiogenesis, rheumatoid arthritic angiogenesis, inflammatory angiogenesis, and the like. The invention also provides a method of inhibiting or preventing restenosis of the lumen of a blood vessel, by repressing angiogenesis from the vaso vasorum, and by promoting large vessel endothelial cell migration to repair the lumen of a large blood vessel. These methods of inhibiting or preventing angiogenesis are provided in vivo and/or in vitro. Also provided are Jagged agonists comprising agents which promote the expression of Jagged, including fibrin and functional derivatives thereof and pharmacologically acceptable chemicals, and xcex3-idiotypic Jagged antibodies.
Moreover, the invention provides a method of promoting or enhancing angiogenesis in a subject comprising administering to a subject in need of such promotion or enhancement a therapeutically effective amount of anti-Jagged or a Jagged antagonist. The angiogenesis being promoted or enhanced comprises wound or injury repair angiogenesis, such as that which occurs in a wound or injury caused by surgery, trauma and/or disease or condition, including diabetes-related wounds or injuries. These methods of promoting or enhancing angiogenesis are provided in vivo and/or in vitro. Also provided are Jagged antagonists comprising Jagged antibodies, anti-sense Jagged, Jagged mutants and pharmacologically acceptable chemicals.
The invention further provides a method for affecting cell differentiation of cells comprising the mesoderm, endoderm, ectoderm and/or neuroderm. Also provided is a method for affecting cell differentiation of cells, wherein the cell types affected comprise hematopoietic stem cells, epithelial cells, vascular smooth muscle cells and dendritic cells.
In addition, the invention provides a pharmaceutical composition used in any of the previously disclosed methods.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.